Background: Pigment epithelium&#8208;derived factor (PEDF), which belongs to the noninhibitory serpin family, has shown the ability to stimulate several physiological processes, such as antiangiogenesis, anti&#8208;inflammation, and antioxidation. In the present study, the effects of PEDF on contractility and calcium handling of rat ventricular myocytes were investigated.

Methods and results: Adult Sprague&#8208;Dawley rat models of acute myocardial infarction (AMI) were surgically established. PEDF&#8208;lentivirus was delivered into the myocardium along and away from the infarction border to overexpress PEDF. Video edge detection was used to measure myocyte shortening in&nbsp;vitro. Intracellular Ca2+ was measured in cells loaded with the Ca2+ sensitive fluorescent indicator, Fura&#8208;2&#8208;acetoxymethyl ester. PEDF local overexpression enhanced cardiac functional reserve in AMI rats and reduced myocardial contracture bordering the infracted area. Exogenous PEDF treatment (10&nbsp;nmol/L) caused a significant decrease in amplitudes of isoproterenol&#8208;stimulated myocyte shortening, Ca2+ transients, and caffeine&#8208;evoked Ca2+ transients in&nbsp;vitro. We then tested a potential role for PEDF receptor&#8208;mediated effects on upregulation of protein kinase C (PKC) and found evidence of signaling through the diacylglycerol/PKC&alpha; pathway. We also confirmed that pretreatment of cardiomyocytes with PEDF exhibited dephosphorylation of phospholamban at Ser16, which could be attenuated with PKC inhibition.

Conclusions: The results suggest that PEDF depresses myocyte contractility by suppressing phosphorylation of phospholamban and Ca2+ transients in a PKC&alpha;&#8208;dependent manner through its receptor, PEDF receptor, therefore improving cardiac functional reserve during AMI.

jah31649-fig-0005: Tracking of contraction changes in neonatal cardiomyocytes. Cardiomyocytes with or without PEDF‐R‐RNAi‐LV or LR‐RNAi‐LV pretransfected were cultured for 24 hours under hypoxia condition. A video edge detection system was used to monitor single‐cell contraction changes. The 4 groups included hypoxia (control), PEDF under hypoxia (PEDF), PEDF with PEDF‐R‐RNAi‐LV under hypoxia (PEDF+siPEDF‐R), and PEDF with LR‐RNAi‐LV under hypoxia (PEDF+siLR). A and C, Amplitude of contraction under normoxia condition. Data are shown as mean±SE from 6 cardiomyocytes (n=6). No significant difference was observed between PEDF and control groups. B, Amplitude of contraction under hypoxia condition. Data are shown as mean±SE from 8 cardiomyocytes (n=8). P<0.001 PEDF versus control; P<0.001 PEDF+siPEDF‐R versus PEDF. D, Statistical analysis of several time points under hypoxia condition. Data are shown as mean±SE from 8 cardiomyocytes (n=8). *P<0.01; #P<0.01. Data are expressed relative to control values measured at the start of each experiment. Results show that PEDF (10 nmol)‐pretreated cardiomyocytes showed a more‐rapid time‐dependent contraction reduction, and the effects could be attenuated by PEDF‐R interference. No significant difference was observed between PEDF and PEDF+siLR groups. LV indicates lentivirus; PEDF, pigment epithelium‐derived factor; PEDF‐R, PEDF receptor.

Mentions:
Cultured rat neonatal cardiomyocytes provide a useful model to understand cardiovascular diseases in vitro on a real‐time cell analysis system, so we further verified the protective effects of PEDF on contractility in neonatal cardiomyocytes. The RNAi vector, PEDF‐R‐RNAi‐LV, of the PEDF‐R gene producing PEDF‐R shRNA and LR were successfully transfected in neonatal cardiomyocytes and were then successfully confirmed by its GFP fluorescence (Figure 4D). Immunostaining for α‐SA showed the establishment of neonatal cardiomyocytes in vitro (Figure 4A through 4C). A video edge detection system was used to monitor the effects of PEDF (10 nmol) on hypoxia‐induced single‐cell contraction changes. Spontaneously beating myocytes added with isoproterenol (1 μm) were exposed to hypoxia for 24 hours with or without PEDF pretreatment. Similarly, PEDF had no significant effect on cell contractility in cultured neonatal cardiomyocytes under normal culture conditions (Figure 5A and 5C). Contractility was then measured during the exposure to hypoxia period (Figure 5B and 5D). All groups’ contractions decreased with the extension of hypoxic time and PEDF pretreatment, showing a more‐rapid time‐dependent trend. PEDF caused a significant decrease in contraction amplitude of spontaneously beating myocytes after 3 hours of hypoxia that persisted throughout the following 8 hours until it was stable. These results clearly demonstrated that PEDF promotes a rapid and significant decrease of contractility in hypoxic neonatal cardiomyocytes, but has no effects on normoxic cardiomyocytes.

jah31649-fig-0005: Tracking of contraction changes in neonatal cardiomyocytes. Cardiomyocytes with or without PEDF‐R‐RNAi‐LV or LR‐RNAi‐LV pretransfected were cultured for 24 hours under hypoxia condition. A video edge detection system was used to monitor single‐cell contraction changes. The 4 groups included hypoxia (control), PEDF under hypoxia (PEDF), PEDF with PEDF‐R‐RNAi‐LV under hypoxia (PEDF+siPEDF‐R), and PEDF with LR‐RNAi‐LV under hypoxia (PEDF+siLR). A and C, Amplitude of contraction under normoxia condition. Data are shown as mean±SE from 6 cardiomyocytes (n=6). No significant difference was observed between PEDF and control groups. B, Amplitude of contraction under hypoxia condition. Data are shown as mean±SE from 8 cardiomyocytes (n=8). P<0.001 PEDF versus control; P<0.001 PEDF+siPEDF‐R versus PEDF. D, Statistical analysis of several time points under hypoxia condition. Data are shown as mean±SE from 8 cardiomyocytes (n=8). *P<0.01; #P<0.01. Data are expressed relative to control values measured at the start of each experiment. Results show that PEDF (10 nmol)‐pretreated cardiomyocytes showed a more‐rapid time‐dependent contraction reduction, and the effects could be attenuated by PEDF‐R interference. No significant difference was observed between PEDF and PEDF+siLR groups. LV indicates lentivirus; PEDF, pigment epithelium‐derived factor; PEDF‐R, PEDF receptor.

Mentions:
Cultured rat neonatal cardiomyocytes provide a useful model to understand cardiovascular diseases in vitro on a real‐time cell analysis system, so we further verified the protective effects of PEDF on contractility in neonatal cardiomyocytes. The RNAi vector, PEDF‐R‐RNAi‐LV, of the PEDF‐R gene producing PEDF‐R shRNA and LR were successfully transfected in neonatal cardiomyocytes and were then successfully confirmed by its GFP fluorescence (Figure 4D). Immunostaining for α‐SA showed the establishment of neonatal cardiomyocytes in vitro (Figure 4A through 4C). A video edge detection system was used to monitor the effects of PEDF (10 nmol) on hypoxia‐induced single‐cell contraction changes. Spontaneously beating myocytes added with isoproterenol (1 μm) were exposed to hypoxia for 24 hours with or without PEDF pretreatment. Similarly, PEDF had no significant effect on cell contractility in cultured neonatal cardiomyocytes under normal culture conditions (Figure 5A and 5C). Contractility was then measured during the exposure to hypoxia period (Figure 5B and 5D). All groups’ contractions decreased with the extension of hypoxic time and PEDF pretreatment, showing a more‐rapid time‐dependent trend. PEDF caused a significant decrease in contraction amplitude of spontaneously beating myocytes after 3 hours of hypoxia that persisted throughout the following 8 hours until it was stable. These results clearly demonstrated that PEDF promotes a rapid and significant decrease of contractility in hypoxic neonatal cardiomyocytes, but has no effects on normoxic cardiomyocytes.

Background: Pigment epithelium&#8208;derived factor (PEDF), which belongs to the noninhibitory serpin family, has shown the ability to stimulate several physiological processes, such as antiangiogenesis, anti&#8208;inflammation, and antioxidation. In the present study, the effects of PEDF on contractility and calcium handling of rat ventricular myocytes were investigated.

Methods and results: Adult Sprague&#8208;Dawley rat models of acute myocardial infarction (AMI) were surgically established. PEDF&#8208;lentivirus was delivered into the myocardium along and away from the infarction border to overexpress PEDF. Video edge detection was used to measure myocyte shortening in&nbsp;vitro. Intracellular Ca2+ was measured in cells loaded with the Ca2+ sensitive fluorescent indicator, Fura&#8208;2&#8208;acetoxymethyl ester. PEDF local overexpression enhanced cardiac functional reserve in AMI rats and reduced myocardial contracture bordering the infracted area. Exogenous PEDF treatment (10&nbsp;nmol/L) caused a significant decrease in amplitudes of isoproterenol&#8208;stimulated myocyte shortening, Ca2+ transients, and caffeine&#8208;evoked Ca2+ transients in&nbsp;vitro. We then tested a potential role for PEDF receptor&#8208;mediated effects on upregulation of protein kinase C (PKC) and found evidence of signaling through the diacylglycerol/PKC&alpha; pathway. We also confirmed that pretreatment of cardiomyocytes with PEDF exhibited dephosphorylation of phospholamban at Ser16, which could be attenuated with PKC inhibition.

Conclusions: The results suggest that PEDF depresses myocyte contractility by suppressing phosphorylation of phospholamban and Ca2+ transients in a PKC&alpha;&#8208;dependent manner through its receptor, PEDF receptor, therefore improving cardiac functional reserve during AMI.